Certain human plasma proteins useful for therapeutic purposes and other applications can be obtained only from pooled blood donations. Recombinant production of plasma proteins is complicated by the fact that these proteins require accurate glycosylation patterns in order to maintain their function and/or half-life in the human body. Therefore even with the attendant risks of viral or other contamination the only approved available source for some proteins such as alpha 1 proteinase inhibitor is human plasma itself.
Alpha-1 proteinase inhibitor (API) is a derivative of human plasma belonging to the family of serine proteinase inhibitors. It is a glycoprotein having an average molecular weight of 50,600 daltons, produced by the liver and secreted into the circulatory system. The protein is a single polypeptide chain, to which several oligosaccharide units are covalently bound. API has a role in controlling tissue destruction by endogenous serine proteinases, and is the most prevalent serine proteinase inhibitor in blood plasma. Among others, API inhibits trypsin, chymotrypsin, various types of elastases, skin collagenase, renin, urokinase and proteases of polymorphonuclear lymphocytes.
API is currently used therapeutically for the treatment of pulmonary emphysema in patients who have a genetic deficiency in API. Purified API has been approved for replacement therapy in these patients. The normal role of API is to regulate the activity of leukocyte elastase, which breaks down foreign proteins present in the lung. When API is not present in sufficient quantities to inhibit elastase activity, the elastase breaks down lung tissue. In time, this imbalance results in chronic lung tissue damage and emphysema.
API was also proposed as a treatment for patients homozygous for the defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR) genes, who suffer from recurrent endobronchial infections and sinusitis, malabsorption due to pancreatic deficiency, obstructive hepatobiliary disease and reduced fertility. The major cause of morbidity and mortality among CF patients is lung diseases. CFTR regulates transport of water and salts in the epithelial cells which cover internal and external surfaces of the body. In CF patients, the CTFR protein is defective due to a mutation, resulting in a defective water and salt transport and the production of thick secretions in several organs (e.g. lung, pancreas).
The membrane defect caused by the CFTR mutation leads to chronic lung inflammation and infection. Chronic lower respiratory infection provokes a persistent inflammatory response in the airway, resulting in chronic obstructive disease. As pulmonary reserves decrease, CF patients become prone to episodes of exacerbation, characterized by worsening symptoms of respiratory infection, particularly by Peusdomonas aeruginosa, accompanied by acute decline in lung function. In normal individuals, elastase secreted by neutrophils in response to infection is neutralized by API, which is known to penetrate into pulmonary tissue. In patients with CF, however, the unregulated inflammatory response overwhelms the normal protease (elastase)/antiprotease (API) balance. The abnormal cycle is destructively self-perpetuating and self-expanding: increased elastase leads to the recruitment of more neutrophils to the lung, which in turn secrete additional proteases. This leads to the accumulation of elastase in the lung and ultimately to tissue damage, destruction of the lung architecture, severe pulmonary dysfunction and, ultimately, death. It is suggested that supplement of additional API may reduce the deleterious effects associated with excessive amounts of elastase. The demand for API already exceeds the availability of the current supply, and this problem may become more pronounced as research suggests additional therapeutic uses for API. In order to maximize the available supply of API, a process for purifying API from human plasma should have the highest yield possible, and alternative sources should be also considered. Therefore, more efficient means of isolation, suitable for GMP (good manufacture practice) large-scale production, is required.
Several groups have reported production of recombinant API. (For example, G. Wright et al., Biotechnology, Vol. 9, pp. 830-834 (1991); A. L. Archibald et al., Proc. Nat'l. Acad. Sci. USA., Vol. 87, pp. 5178-5182 (1990)). However, at present, human plasma is the only approved source of therapeutic API.
Various methods of purifying API from human plasma have been described. The majority of these methods are directed to laboratory scale isolation while others pertain to production on a commercial level. Several methods of isolation are disclosed, for example in U.S. Pat. Nos. 4,379,087 and 5,610,285. Many early methods employed ammonium sulfate precipitation from human plasma followed by dialysis, further employing chromatographic step on DEAE-cellulose. However, the methods described for dialysis are not easily applicable to large-scale purification, and are lengthy, time-consuming processes likely to compromise the activity of the isolated protein.
A large-scale purification of API from human plasma was disclosed by Kress et al., (Preparative Biochem., 3:541-552, 1973)). The precipitate from the 80% ammonium sulfate treatment of human plasma was dialyzed and chromatographed on DEAE-cellulose. The concentrate obtained was again dialyzed and gel filtered on SEPHADEX™ G-100. The API-containing fractions were chromatographed twice on DE-52 cellulose to give API.
Glaser et al., (Preparative Biochem., 5:333-348, 1975) isolated API from Cohn Fraction IV-1 paste. In this method, dissolved IV-1 fraction was chromatographed on DEAE-cellulose, QAE-SEPHADEX™, concanavalin-A-SEPHAROSE™, and SEPHADEX™-G-150 to give API. However, Glaser et al. achieved only a 30% overall yield from fraction IV-1 paste.
Podiarene et al., (Vopr. Med. Khim. 35:96-99, 1989) reported a single step procedure for isolation of API from human plasma using affinity chromatography with monoclonal antibodies. API specific activity was increased 61.1 fold with a yield of only 20% from plasma.
Burnouf et al., (Vox. Sang. 52:291-297, 1987) starting with Cohn Fraction effluent II+III used DEAE chromatography and size exclusion chromatography to produce an API 80-90% pure (by SDS-PAGE) with a recovery of 65-70% from this effluent.
Hein et al., (Eur. Respir. J. 9:16s-20s, 1990) presented a process that employs Cohn Fraction IV-1 paste as the starting material and utilized fractional precipitation with polyethylene glycol followed by anion exchange chromatography on DEAE-Sepharose™. The final product has a purity of about 60% with 45% yield from IV-1 paste.
Dubin et al., (Prep. Biochem. 20:63-70, 1990) used a two-step chromatographic purification whereby alpha-PI, C1-inhibitor, alpha-1 antichymotrypsin, and alpha-1 trypsin inhibitor were first eluted from Blue Sepharose™ and then API was purified by gel filtration. Purity and yield data were not given.
U.S. Pat. No. 4,749,783 discloses a method where biologically inactive proteins in a preparation were removed by affinity chromatography after a viral inactivation step. The basis of the separation between the native and denatured forms of the protein was the biological activity of the native protein towards the affinity resin and not physical differences between the native and denatured proteins.
An integrated plasma fractionation system based on polyethylene glycol (PEG) was disclosed by Hao et al. (Proceedings of the International Workshop on Technology for Protein Separation and Improvement of Blood Plasma Fractionation, Sep. 7-9, 1977, Reston, Va.). In the published method Cohn cryoprecipitate was mixed with increasing concentrations of PEG in order to obtain four different PEG fractions. The four fractions obtained were 0-4% PEG precipitate, 4-10% PEG precipitate, 10-20% PEG precipitate and 20% PEG supernatant. The 20% PEG supernatant fraction was dominated by albumin but also contained most of the API. However, this fraction also contained numerous other proteins, including all of the alpha-1-acid glycoprotein, antithrombin III, ceruloplasmin, haptoglobin, transferrin, Cl esterase inhibitor, prealbumin, retinol binding protein, transcortin, and angiotensinogen.
Several other groups have combined PEG precipitation with other purification methods in an attempt to isolate API. For instance, U.S. Pat. Nos. 4,379,087; 4,439,358; 4,697,003 and 4,656,254, all employ a PEG precipitation step in processes of isolating API. However, the disclosed methods do not attempt to separate active from non-active API.
Japanese Patent No. 8-99999 discloses the use of PEG precipitation in combination with an SP-cation exchanger. The methods described therein do not separate fully active API from inactive API. The specific activity of fully active API should be 1.88 (using an Extinction coefficient 5.3), but the product achieved by this process only shows a relative activity of 1.0. Moreover, the best yield achieved by combining PEG precipitation and SP-cation exchange steps was only 50%, and does not appear to be easily scaled up to a commercial production level.
U.S. Pat. No. 5,610,285 discloses a purification process which combines successive anion and cation exchange chromatography steps. The initial anion exchange chromatography step binds API to the column; however, it also binds numerous contaminating proteins, particularly lipoproteins. Lipoproteins are plentiful in many of the materials from which API is isolated (e.g. Cohn IV-1 paste), and so tend to occlude the column. Such occlusion requires columns of considerable size, additional dialysis/filtration steps, and at least two cation chromatography steps. Those requirements reduce efficiency and practicality of the method for large-scale processes. Further, in the '285 process all API, both inactive and active protein, bind to the anion exchange column. When the API is eluted from that column in accordance with that method, i.e., high salt phosphate buffer, both active and inactive protein come off the column. Thus, there is no separation of the active from the inactive protein.
U.S. Pat. No. 6,093,804 discloses a method combining removal of lipoproteins from the source material, followed by subsequent anion and cation exchange steps, which result in highly purified, highly active API. However, this method proved to be efficient for small to mid-scale production of processing source material in the range of few kilograms.
As mentioned above, the demand for API exceeds available supply. Thus, there is a great need for, and it would be highly advantageous to have a process for a large-scale production of API, in which quality, which refers to both purity and activity, is not compromised for quantity. Moreover, it would be highly beneficial to have stable, viral-inactivated ready to use formulations of the purified API.